Molecular chaperones are present in all organisms and are highly conserved. Chaperones participate in many and perhaps all cellular processes. They function during non-stress conditions to facilitate folding of newly synthesized proteins and to refold or target for degradation abnormal or misfolded proteins. During cell stress, chaperones play an essential role in preventing the appearance of folding intermediates that lead to irreversibly damaged and aggregated proteins. They promote recovery from stress by disaggregating and reactivating proteins and by delivering damaged proteins to compartmentalized proteases. Protein aggregation and misfolding are primary contributors to a diversity of human diseases, including Alzheimers, Parkinsons, type II diabetes, cystic fibrosis, and prion diseases. Understanding how chaperones function and how they interact with proteases will provide the foundation for discovering cures and preventions for the devastating diseases caused by protein misfolding, premature degradation, and formation of toxic protein aggregates. Our previous work showed that Escherichia coli ClpA, an AAA+ protein and the regulatory component of ClpAP protease, has molecular chaperone activity. This work and the work of others demonstrated that Clp ATPases comprise a family of ATP-dependent chaperones. Two members of the Clp family, ClpB of prokaryotes and Hsp104 of yeast, collaborate with Hsp70/DnaK and Hsp40/DnaJ to rescue aggregated proteins. However, the mechanisms that elicit and govern the protein remodeling activities of ClpB/Hsp104 remain unclear. Recently we discovered that ClpB and Hsp104 possess innate protein remodeling activities. Unexpectedly, mixtures of ATP and ATP&#947;S (a non-physiological and slowly hydrolyzed ATP analog) were found to elicit intrinsic activation, disaggregation, and unfolding activities of ClpB and Hsp104. Impairing hydrolysis by mutations at individual nucleotide binding sites of ClpB and Hsp104 similarly elicits remodeling activities, implying that asymmetric ATP hydrolysis evokes the innate chaperone functions. Because ClpB/Hsp104 acts in conjunction with the DnaK/Hsp70 system in vivo, these results suggested that the role of the DnaK/Hsp70 chaperone system is to modulate the ATPase of ClpB/Hsp104. However our recent results show that ClpB and DnaK function synergistically and thus ATP&#947;S does not simply mimic the function of the DnaK/Hsp70 system. We have also been investigating how another Clp protein, ClpX, acts in proteolysis with a proteolytic component, ClpP, and an adaptor protein, RssB. RssB is required to specifically promote degradation of the stationary phase RNA polymerase sigma factor, sigma S, by ClpXP. Results obtained from the analysis of RssB mutants and ClpX mutants are revealing the mechanism of action of RssB in the regulation of sigma S stability. We are currently studying the E. coli DnaK chaperone and its two co-chaperones, DnaJ and GrpE. CbpA is a DnaJ homolog that is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. The cbpA gene is in an operon with an open reading frame, referred to as cbpM, for modulator of CbpA. We have shown that in vitro CbpA forms a physical complex with CbpM. The consequence of this interaction is the inhibition of both the DnaJ-like co-chaperone activity and the DNA binding activity of CbpA. We have found that in vivo cbpA and cbpM are co-transcribed, with maximal expression occurring at the transition from exponential growth to stationary phase. In addition, the level of CbpA is similar to that of CbpM and the two proteins interact physically in vivo throughout the growth cycle. Furthermore, we demonstrated that this interaction results in the inhibition of the co-chaperone activity of CbpA. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by the expression of an inhibitor specific for one of the components